The objective of this proposal is to study the molecular mechanisms by which acetyicholine (ACh) released from olivocochtear efferents influences cochlear outer hair cells (OHCs). The nicotinic ACh, receptor (nAChR) on OHCs contains the a9 nAChR subunit, and may also contain the newly discovered al0 nAChR. At least two hypotheses have been proposed concerning the contribution cochlear efferents make to hearing: 1) they protect the cochlea from acoustic overstimulation, and 2) they enhance sound recognition in the presence of background noises. In the last project period we investigated the first hypothesis by devising a test of the cochlear efferent reflex strength and found that reflex strength is positively correlated to the expression of a9 nAChR. Furthermore, we discovered that animals with stronger efferent reflexes exhibited less cochlear damage upon noise exposure. The proposed experiments will extend those studies to test whether a9 and al0 subunits of the nAChR form a heteromeric complex and whether modulating expression of this complex in adulthood can influence recovery of cochlear function after acoustic overstimulation. The first specific aim is to determine if a9and alO nAChRs form functional heteromeric receptors in cochlear hair cells in vivo using coimmunoprecipitation and immunohistochemical techniques. The second specific aim will determine if upregulation (or downregulation) of a9, al0, or a9 + alO nAChR expression in adult guinea pig hair cells strengthens (or weakens) the efferent reflex and reduces (or increases) susceptibility to noise damage. These experiments will use a modified adenovirus to deliver a9 and a10 overexpression, dominant negative, and antisense constructs mRNAs directly to the scala tympani of adult guinea pigs. Control studies performed in Xenopus oocytes will be used to validate the receptor coimmunoprecipitation techniques and to verify that dominant-negative and antisense receptor constructs inhibit responses to ACh. Information gained from these studies will contribute to future drug therapies that target cholinergic receptors of the cochlear efferent system with the goal of diminishing noise-induced hearing loss in humans.